Diamond-Blackfan anemia (DBA) is the first human disease known to be caused by mutations in ribosomal protein (RP) genes. It is characterized by anemia, physical anomalies, and increased risk of malignancy. To date mutations in nine RP genes, RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, RPS7, RPS10 and RPS26 have been reported in ~53% of DBA patients. Recently, we finished screening all 80 ribosomal protein genes and have obtained evidence for mutations in two additional RP genes, RPL19 and RPL26, in ~2% of DBA patients. Despite this significant progress in deciphering the genetic causes of DBA, diagnostics and genetic counseling for these patients are still severely hampered by the fact that roughly 50% of cases have mutations in genes yet to be identified. This lack of confirmed genetic etiology also makes it difficult to create suitable animal model for DBA and to perform further studies on the mechanisms of the disease. To address this issue, we are performing comparative genomic hybridization on 150 DNA samples from DBA probands without known mutations to search for deletions and duplications in the 80 RP genes we have already sequenced. We also propose to perform whole exome sequencing ("next generation" sequencing) on 28 DNA samples from DBA probands who were screened for all 80 RP genes and are negative for mutations in these genes. Comparative genomic hybridization will allow us to identify microdeletions or duplications in RP and other candidate genes;the whole exome sequencing (sequencing of all exons and intron-exon boundaries) will allow us to identify the additional DBA genes, which we hypothesize will likely encode proteins involved in ribosomal biogenesis or function. To further test our hypothesis that abnormal ribosomal biogenesis underlies the mechanism of DBA in all patients, we will also perform pre-rRNA maturation assays on RNA samples from lymphoblastoid cell lines from patients with mutations in the newly discovered genes. Our hypothesis predicts that abnormal maturation of pre-rRNA will be a common feature in patients with newly discovered genes. We also hypothesize that profound clinical heterogeneity in DBA is a consequence of genetic variants that influence the presence of associated congenital birth defects or patients'response to steroid treatment. Investigating the influence of these variants would expand our understanding of the mechanism of DBA and potentially, especially for the association with response to steroids, could be a platform for the targeted therapies for DBA. The following three specific aims will allow us to find the genetic basis of DBA and the molecular mechanism of anemia in this disease. Specific Aim 1. Identify the novel gene(s) causing DBA in about 50% of patients without known mutations. Specific Aim 2. Determine the role of DBA genes in pre-RNA maturation. Specific Aim 3. Identify modifier genes in DBA by performing a genome-wide association study. PUBLIC HEALTH RELEVANCE: Diamond-Blackfan anemia is the first human disease known to be caused by mutations in ribosomal protein genes. It is characterized by anemia, physical anomalies, and increased risk of malignancy. The treatment of choice is a steroid therapy, but this has significant side effects. In order to find better treatments for these patients it is important to know the genetic basis and understand the mechanism of the disease. The goal of this project, is to identify genes in approximately 50% of patients without known mutations and to explore the role of ribosomal proteins in the pathogenesis of Diamond-Blackfan anemia, which will establish the basis for better treatment discovery.